Monitoring in vivo concentrations of physiologically relevant compounds to improve diagnosis and treatment of various diseases and disorders is a desirable goal and would enhance the lives of many individuals. Advances in this area show particular promise in the area of facilitating adequate metabolic control in diabetics. Currently, most diabetics use the “finger stick” method to monitor blood glucose level, and patient compliance is problematic due to pain caused by frequent finger sticks. As a consequence, there have been efforts to develop non-invasive or minimally invasive in vivo and more efficient in vitro methods for frequent and/or continuous monitoring of glucose in the blood or other biological fluids.
The approaches to frequent and/or continuous in vivo monitoring tend to fall into two general categories: “non-invasive” and “minimally invasive.” Noninvasive monitoring determines analyte levels by directly tracking spectroscopic changes in skin and tissue. Infrared radiation and radio wave impedance spectroscopy are examples of this technology. Progress with these approaches has been slow due to the requirement for frequent calibration, reproducible sample illumination, and variances in spectroscopic backgrounds between individuals. The “minimally invasive” approach avoids direct extraction of blood from the body and relies on the monitoring of signal changes in biological fluids using an intermediate sensing element. Biosensors of this type are devices capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element that is combined with a transducing (detecting) element.
Most conventional systems for frequent or continuous analyte monitoring involve amperometric biosensors that employ enzymes such as glucose oxidase (GOx) to oxidize glucose to glucuronic acid and hydrogen peroxide, generating an electrochemical signal. These sensors are subject to inaccurate measurement due to oxygen deficiency and buildup of oxidation by-products. An accurate measurement of glucose concentrations requires an excess of oxygen, which is generally not present in human blood or interstitial fluid. Also, the electrochemical reaction itself generates a buildup of oxidation byproducts that may inhibit and degrade both the enzyme and its protective layer.
Biosensors based on optical rather than electrochemical signals have also been developed and may offer significant improvements in stability and calibration. For example, referencing an analyte-dependent optical signal against a second analyte-independent signal can correct for sources of noise and instability in the sensor. However, the potential of optical sensing for in vivo analyte detection has not yet been realized. One reason for this is that many current optical sensing methods rely on enzymatic chemistry such as glucose oxidase. In one common method, an oxygen-sensitive fluorescent dye is used to monitor the consumption of oxygen by the GOx enzymatic reaction. Although this is an optical biosensor, with the fluorescence signal level varying with changing oxygen levels, such a sensor is subject to the same problems as amperometric devices based on this same chemistry: oxygen deficiency and enzyme degradation.
To overcome the challenges associated with enzyme sensing (e.g., GOx), whether electrochemical or optical, non-enzymatic protein-based optical or fluorescent sensing is being explored. Labeled concanavalin A and dextran have been used to create a competitive FRET assay; however, this system requires entrapment of both components, and the dynamic range of the assay is limited. See Ballerstadt, R., Schultz, J. S.; “Competitive-binding assay method based on fluorescence quenching of ligands held in close proximity by a multivalent receptor.” Anal. Chem. Acta 345 (1-3): 203-212 (1997). See also, Russell, R. J., Pishko M. V., Gefrides C. C., McShane, M. J., Cote, G. L.; “A fluorescence-based glucose biosensor using concanavalin A and dextran encapsulated in a poly(ethylene glycol) hydrogel” Anal. Chem. 71 (15): 3126-3132 (1999).
Another protein-based sensing chemistry uses the Esherichia coli (E. coli) periplasmic receptor, glucose-galactose binding protein (GGBP) to generate a fluorescence signal in response to glucose binding. See, for example, Tolosa, L., I. Gryczynski, L. R. Eichhorn, J. D. Dattelbaum, F. N. Castellano, G. Rao, and J. R. Lakowicz; “Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein” Anal. Biochem. 267: 114-120 (1999); Hellinga, H. W., and J. S. Marvin; “Protein engineering and the development of generic biosensors.” Trends Biotechnol. 16: 183-189 (1998); Salins, L. L., R. A. Ware, C. M. Ensor, and S. Daunert, “A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein” Anal Biochem 294: 19-26 (2001); and de Lorimier, R. M., J. J. Smith, M. A. Dwyer, L. L. Looger, K. M. Sali, C. D. Paavola, S. S. Rizk, S. Sadigov, D. W. Conrad, L. Loew, and H. W. Hellinga. “Construction of a fluorescent biosensor family” Protein Sci. 11: 2655-2675 (2002). GGBP undergoes a substantial conformation change upon ligand binding, trapping the ligand between its two globular domains. See, for example, Shilton, B. H., M. M. Flocco, M. Nilsson, and S. L. Mowbray; “Conformational changes of three periplasmic receptors for bacterial chemotaxis and transport: the maltose-, glucose/galactose- and ribosebinding proteins” J. Mol. Biol. 264: 350-363 (1996). By site-specifically labeling the protein with an environmentally sensitive fluorophore this attribute can be exploited to generate a fluorescent signal. See, for example, Salins, L. L., R. A. Ware, C. M. Ensor, and S. Daunert; “A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein” Anal Biochem 294: 19-26 (2001). Because GGBP neither consumes glucose nor generates reaction products, it can be used as a reagentless sensor. This may provide greater accuracy and reliability than amperometric biosensors.
While a number of groups have developed GGBP mutations capable of responding to glucose in the physiological range, there have been no reports of a functional biosensor device based on binding protein technology that is suitable for in vivo or in vitro analyte monitoring. A functional frequent and/or continuous biosensor must couple the sensing element to the optical sensing elements while maintaining sensor integrity and functionality as well as patient comfort. For example, the biological recognition element and accompanying transducing element should preferably be incorporated within biocompatible material that shields the sensing element from the immune system, permits analyte diffusion in and out, and avoids leaching of the sensing element into the patient blood or other biological fluid (e.g., interstitial fluid). Since binding proteins require orientational control and conformational freedom to enable effective use, many physical absorption and random or bulk covalent surface attachment or immobilization strategies as taught in the literature generally are either suboptimal or unsuccessful. Further, a means for interrogating the sample with light in a reproducible and/or controlled fashion must be devised.
One approach generally known is to couple the sensing element to one end of an optical fiber and to couple the optical elements such as excitation sources or detectors to the other end. However, coupling of binding proteins to one end of an optical fiber is subject to the above-mentioned challenge of preserving conformational and/or orientational mobility of the protein. In addition, fiber optic cabling is often impractical from a patient-use point of view since patients may need to remove or replace the sensor periodically. Replacement of the entire fiber can be costly and inconvenient. Finally, the optical system, comprising, e.g., excitation sources, detectors, and other optical elements must be sufficiently robust to tolerate or correct for changes in optical alignment due, for example, to patient motion or drift of the electronics in the optical reader. The optical system must also be sufficiently sensitive to detect signal from reporter dyes without relying on high power consumption and/or large-sized elements that would render the system unportable and hence unwearable.
Accordingly, there is a need for a biosensor that incorporates in its sensing element a binding protein with conformational and/or orientational mobility coupled to optical sensing elements that provide a wearable and robust device.